Comparative hemopoietic and growth factor responses to vitamin B12, folic acid, and iron*†

Comparative Hemopoietic and Growth Factor Responses to Vitamin B,,, Folic Acid, and Iron*J By ERSILIA F. DE GUIA and MORRIS M. WOLFREDS Experimental data have been presented on the effectiveness of vitamin B12,folic acid, and iron on anemia in chicks produced by a basal deficient diet. Hemopoietic and growth factor values for combinations of folic acid, vitamin Biz, and iron have been demonstrated in varying dosages and modes of administration. Results illustrate the rate of recovery, growth, and effectiveness of the supplements. EXPERIMENTAL evidence has indicated a further need for testing the effectiveness of Day-old white Leghorn chicks with numbered folic acid and vitamin B12 on anemic chicks, and rings on their legs for identification were placed in for additional information concerning the hemo- electrically heated cages. The chicks were given a poietic value of combinations of folic acid and . special prepared diet deficient in folic acid and were vitamin B12with iron; no previous work has been allowed to indulge in the special feed and water ad libitum until they became anemic. Blood samples presented using such combinations. I n addi- were taken periodically by wing vein punctures, and tion, attempts were made to correlate the growth red blood cell counts and hemoglobin levels were recorded. factor of folk acid and vitamin B12with iron. The folic acid-deficient basal ration consisted of Gibson and Howard (I), in 1923, demonstrated the following formula: that a diet high in iron content produced a favorable influence in pernicious anemia. Elvehjem Casein . . . . . . . . . . . . . . . . . . . . . . . . 25.0 Gm. and Hart ( 2 ) , in 1929, reported that day-old ... 10.0 Gm. chicks placed on a diet of cow’s milk together with polished rice, calcium carbonate, and sodium ............... 5.0 Gm. Methionine. . . . . . . . . . . . . chloride became anemic. The addition of ferrous Cod liver oil.. . . . . . . . . . . . . . . . . . 2,000.0 mg. sulfate to the ration stimulated hemoglobin synthesis within a short period of time. Hogan Riboflavin . . . . . . . . . . . . . . . . . . . . 0.8 mg. 5.0 mg. Nicotinic acid. . . . . . . . . . . . . . . . . and Parrott (3), in 1940, observed that day-old 0.6 mg. Pyridoxine. . . . . . . . . . . . . . . . . . . . chicks fed a folic acid-deficient diet developed 2.0 mg. Calcium pantothenate. . . . . . . . . . Biotin . . . . . . . . . . . . . . . . . . . . . . . . 0.02 mg. anemia and failed to grow. Nichol, Dietrich, ....... 200.0 mg. Elvehjem, and Hart (4),in 1949, observed that ....... Inositol. . . . . . . . 100.00 mg. a folic acid deficiency in chicks fed a purified raa-Tocopherol. . . . . . . . . . . . . . . . . . 0.30 mg. 2.50 mg. tion was accentuated by supplying vitamin BIZ. Vitamin K I . .. . . . . . . . . . . . . . . . . . Nichol, Harper, and Elvehjem (5), in 1949, inBlood counts and hemoglobin levels were taken duced severe anemia in chicks by intramuscular at weekly intervals until the red blood cell count injections of phenylhydrazine hydrochloride reached below two million/cu. mm. of blood and the after a depletion period on a folk acid-deficient hemoglobin level was 5 Gm. or less per 100 cc. of ration. Pure vitamin Blz completely replaced blood. This has been previously determined as liver extract in stimulating the formation of hemo- the anemic level in chicks (3). Twenty-four to thirty days were usually required to reach the globin in the presence of folic acid. Schaefer, anemic level. Salmon, Strength, and Copeland (6), in 1950, reOf 180 chicks given a folic acid-deficient diet, 132 ported that vitamin Blz could replace folic acid survived the diet to reach the anemic level, indiunder their experimental conditions ; however, cating a mortality of 26.6%. The chicks affected slightly higher hemoglobin levels were consist- by the deficiency showed body tremors. extended wings with inability to maintain balance, and signs ently observed when both nutrients were present of paralysis. They remained in this condition two in the diet. Vitamin BIZhas been shown to be a to three days before dying. All chicks showed poor growth factor for rats (7), pigs (8), and chicks feathering, poor growth, enlarged crops, soft stools, and anorexia. (9, 10). Once anemic, the chicks were divided into five main groups according to their mode and manner of *Received November 21 1950 from the University of Southern California, Schooi of Pharmacy, Department of treatment. They were weighed at the peak of Pharmacology. t This paper is based on a thesis resented by Ersilia F. their anemia and their weights were recorded XPERIMENTAL

De Gtua t o the Graduate School of t i e University of Southern California, in partial fulfillment of the requirements for the degree of Master of Science in Pharmacy. $ Assistant Professor of Pharmacology. the University of Southern California. School of Pharmacy, Los Angeles.

1 Salts used according to nutritional requirements, J . Nutrition. 32. 460(1946L

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(Doses:Vitamin BIZ,6 pg.; folk acid, 6 mg.; ferric ammonium citrate, 6 mg.) A, 6 pg. vitamin BIZ; B , 6 mg. folic acid; c, 6 pg. vitamin Biz plus 6 mg. folic acid; D, 6 pg. vitamin BU plus 6 mg. ferric ammonium citrate; E, 6 mg. folic acid plus 6 mg. ferric ammonium citrate. weekly during their process of treatment. On the basis of dosage and manner of administration, each main group was divided into four subgroups, using three chicks as a set of controls for each subgroup. Upon treatment, blood counts and hemoglobin levels were taken a t one- t o two-day intervals (see Fig. 1).

Leghorn chicks ranged from 11.1 t o 12.8 Grn./100 cc. of blood. Vitamin BIs.-Vitamin Bu in 6- or 3-pg. doses administered either parenterally or orally did not cause a significant improvement on the red blood cell counts and hemoglobin levels of anemic chicks. Vitamin B1* given parenterally in doses of 6 pg. produced greater growth in the chicks than did 3 DISCUSSION pg. of vitamin B1z/100 Gm. of diet. All the chicks According t o Doyle, Matthews, and Roberts ( l l ) , which were given 3 pg. of vitamin B1p/100 Gm. of chicks in confinement have red cell counts ranging diet died within ten days after treatment was begun. from 2.0 t o 2.3 millions/cu. mm. Kelly and According t o Charkey, et al. (14), vitamin B12 functions by enhancing the utilization of the circulating Dearstyne (12) have reported that the number of red blood cells in confined chicks ranged from 2.02 amino acids for the building of fixed tissues and a t o 3.49 millions/cu. mm.; in the majority of analy- deficiency of this vitamin will result in slow growth. No improvement in the feathering of the chicks ses the counts fell between 2.27 and 3.01 millions/ cu. mm. Cook (13) has presented counts made by was noted. Folid Acid.-The chicks given daily doses of 6 three investigators, as well as by himself, and concluded that the average red cell count of 2.5 millions/ mg. of folk acid parenterally showed rises in red cu. mm. was a normal for twenty-six-day old t o blood cell counts and hemoglobin levels up to the eighty-six-day old chicks. However, in our experi- twentieth day of treatment, but folic acid was not ments the normal number of red blood cells ranged sufficient t o cause a return t o normal. Six millifrom 3.73 to 4.71 millions/cu. mm. for week-old grams of folic acid was found t o be more active orally white Leghorn chicks. This was found to be higher and parenterally than 3 mg. Chicks treated with than the red cell counts reported for older chicks folic acid showed a greater increase in weight than those which received vitamin B12 as the supplement, in the above literature. Cook (13) also found that the average normal thus confirming the work of Williams, Nichol, and Normal feathering developed hemoglobin level for twenty-six-day old to one- Elvehjem (15). hundred-thiiy-eight-day old chicks was 10.2 within four days. All of the chicks survived. Folid Acid Plus Vitamin Blz.-Chicks treated Gm./100 cc. of blood. A hemoglobin level higher than this value was obtained when casein was used with a combination of folic acid and vitamin BU as the principal source of protein in the diet. The showed the greatest increase in weight. Blood results of our experiments demonstrate that the counts and hemoglobin levels of chicks treated with normal hemoglobin levels for seven-day old white 6 pg. of vitamin Bl2 plus 6 mg. folk acid returned to

June, 1951

increases in weight than chicks treated with vitamin BIZ, folic acid, and vitamin B1, plus iron. This combination also regenerated more red blood cells and higher hemoglobin levels than folic acid alone, hut was not sufficient to bring them back t o normal. Normal feathering began t o develop within four days. Controls.-Three chicks were used as controls for each supplement, and it was noted t h a t the control chicks consumed less food than the treated anemic chicks; however, the increase in weight was not in proportion to the amount of food ingested. Chicks which received the supplements rapidly became more active than the controls.

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Fig. 2.-Effects of various supplements on the growth factor when administered parenterally. (Doses: vitamin BIZ,6 pg. ; folic acid, 6 mg. ; ferric ammonium citrate, 6 mg.) A, 6 a.vitamin BIZ,B , 6 mg. folk acid; C, 6 pg. vitamin Blz plus 6 mg. folic acid; D,6 pg. vitamin B12plus 6 mg. ferric ammonium citrate; E, 6 mg. folic acid plus 6 mg. ferric ammonium citrate.

normal after nine daily injections. This dosage also produced the greatest increase in weight. Normal feathering began t o develop within two days. Evidence indicates that vitamin Blz may function in an analogous manner to a catalyst by facilitating the uptake of folic acid by the tissues of the body, or vice versa. Vitamin BIZPlus Iron.-Chicks treated with vitamin Blz plus iron showed slightly greater increases in weight than those treated with vitamin BIZalone. Three milligrams of ferric ammonium citrate plus 3 pg. of vitamin B12 given parenterally proved less active than 3 pg. of vitamin Blz administered alone in the same way. However, 6 pg. of vitamin BIZ plus 6 mg. ferric ammonium citrate produced a hemopoietic response comparable to that produced by 6 mg. of folic acid plus 6 mg. of ferric ammonium citrate. This was also observed in the case of oral administration where ferrous gluconate was used instead of ferric ammonium citrate. No improvement in the feathering of chicks was noted. Folid Acid Plus Iron.--Chicks treated with folk acid and iron parenterally or orally showed greater

Of all the supplements administered, only the combination of folic acid and vitamin BI2 actually cured the induced anemia, although other hemopoietic agents also influenced the regeneration of red blood cells and hemoglobin. Blood counts and hemoglobin levels returned to normal in twelve days following oral administration and i n nine days following parenteral administration. Folic acid or vitamin Bl2 when combined with iron produced greater red blood cell a n d hemoglobin regenerations than either one used alone. In t h e decreasing order of their effectiveness on t h e growth factor, orally and sparenterally, the supplements were found as follows: (a) folic acid plus vitamin B12 (b) folic acid plus iron, (c) folic acid, (d) vitamin BIZ plus iron, and (e) vitamin &. REFERENCES (1) Gibson R. B.. and Howard, C. P., Arch. Znfcrnnl Med.. 32, l(1953). (2) Elvehjem, C.A.. and Hart, E. B., J . B i d . Chcm., 84. 131(1929). (3) Hogan, A. G.. and Parrott, E. M., ibid., 132. 507 ( 1940). (4) Nichol C. A. Dietrich L. S. Elvehjem, C. A.. and Hart, E. B., J: Nuiriiion, 39, 287 (igig). (5) Nichol C A Harper A. E. and Elvehjem, C. A,, Proc. SOC.E x $ f l . ' B d i . Mcd., $1, 34(i949). (6) Schaefer E A. Salmon W. D. Strength, D. K.. and Copeland, D. H., J : Nufrifidn,40,95?1950). (7) Zucker. L. M.. and Zucker. F. M.. Arch. Biochem.. 16. 115(1948). (8) Johnson, B. C., and Neumann, A. L., J . Biol. Chem., 178, 1001(1949). (9) Nichol C. A. Dietrich L S. Cravens W. W. and Elvehjem C. 'A. Prdc. SOC.E i g f l ' B h . Mcd. '70. 40(i949). (10) Oit, W.k,Rickes, E. L..'and Wood, t.R., J . B i d . Chem. 174, 1047(1948). (11)' Doyle, L. P.,Matthews. F. P., and Roberts, R. E . . Poulfrr Scr. 9 6(1929) (12) Kelfy.'J. W.,and Dearstyne, R. S., N . C. Agric. E t p f l . S f o f .Bull. 50 1935. (13) Cook, S. F.. 'Poulfry,Sci,. 16, 291(1937). (14) Charkey. L. W.,Wilgus. H . S., Patton, A. R.. and E r p f t . Biol. Med., 73, 21(1950). Gassner, P. X . , Proc. SOC. (15) Williams, J. N.,Jr.. Nichol, C. A,, and Blvehlem, C. A., J . B i d . Chem., 180 ,689(1949).